Role of N2-fixation in the sustainability of the ponded grass pasture system

Soil Bid.

Pergamon

0038-0717(94)00221-5

Biochem.

Vol. 27, No. 415, pp. 441-445, 1995 Copyright 0 1995 Elswier Science Ltd Printed in Great Britain. All ri&s nsewcd 0038-0717/95 s9.50 + 0.00

ROLE OF NrFIXATION IN THE SUSTAINABILITY PONDED GRASS PASTURE SYSTEM

OF THE

K. L. WEIER,‘* P. A. PITTAWAY* and J. H. WILDIN’ CSIRO, Division of Tropical Crops and Pastures, 306 Carmody Road, St Lucia, Brisbane, Qld 4067, Australia, ZDepartment of Plant Production, The University of Queensland, Gatton College, Lawes, Qld 4343, Australia and ‘Department of Primary Industries, Box 6014, Rockhampton Mail Centre, Rockhampton, Qld 4702, Australia Summary-Ponded pastures in northern Australia produce green fodder in the seasonally dry winter period and may be employed to reduce grazing pressure on dryland pastures. The soils under ponded pasture, currently 26,000 ha in Queensland, are inherently infertile. This study was conducted to determine if a non-symbiotic association between bacteria and grass roots was responsible for the supply of N to ponded grasses. Intact soil-plant cores were obtained from a new ponded pasture of Aleman (Echinochloa polystachya)and an I-year-old pasture of Hymenachne (Hymenachneamplexicaulis).Nitrogenase (Nr-ase) activity was measured using the acetylene reduction assay and bacteria were selectively isolated to N-free malate medium from root segments of the most active plants. N2-ase activity of the intact soil-plant cores ranged from 76 to 380 g N ha-’ d-’ for the Aleman pasture and from 5 to 179 g N ha-’ d-i for the Hymenachne pasture. Assays on excised roots showed the greatest activity on adventitious roots formed on the submerged nodes of Hymenachne stems. No major differences in colony morphology were detected in Nz-fixing bacteria isolated from the roots of the two grasses. An association appeared to exist between bacteria and the roots of both grasses with most of the N for the young Aleman pasture being fixed N, whereas the tixed N supply for the older Hymenachne pasture was supplemented by the mineralization of organic N.

INTRODUCMON

The use of ponded pasture systems for maintaining cattle liveweight gain in the dry season (May-November) is gaining favour in northern Australia because of the provision of high quality green forage to supplement the native pasture (Wildin, 1993). Excess water or run-off from wet season rainfall is stored in bays and used to grow grasses such as Para (Brachiaria mutica), Aleman (Echinochloa polystachya) and Hymenachne (Hymenachne amplexicaulis), all of which are capable of growth although roots are fully submerged. Increased productivity from inherently infertile soils after an initial application of only 125 kg diammonium phosphate (DAP) ha - I during establishment (Wildin and Chapman, 1988) may be due to non-symbiotic N2-fixation, increased mineralization of N in the older pond or a combination of both (Weier and Pittaway, 1993). Biological N2-fixation occurs in tropical grass production systems through bacteria growing in association with roots (Dobereiner et al., 1972a; Weier and MacRae, 1980) and contributes to the N balance of some rice crops (App et al., 1984). However, information is scarce on the nutrient processes in ponded pastures despite similarities between this system and flooded soil production systems such as rice (Buresh et al., 1980). The aims of our study were (1) to establish if increased productivity *Author for correspondence.

was due to N&ation associated with the grass roots, (2) to ascertain if organic N was released more quickly under flooded conditions and (3) to isolate and identify diazotrophic bacteria associated with the roots of the most active soil-plant cores. MATERULS AND METHODS Sites

The two experimental ponds formed part of larger ponded pasture systems, one located at Bear’s Lagoon, Moura (24”34’S., 149”59’E.) and the other at Granite Vale, St. Lawrence (22”37’S., 149”3O’E.). Both regions have a humid, tropical climate with a mean annual rainfall of 707 and 977 mm, respectively. At Bear’s Lagoon, the ponds were planted to Aleman in mid- 1992 with water being pumped from the Dawson River into the bays to establish a controlled ponding system. At Granite Vale, ponds of Hymenachne had been established for 8 years and were filled with water collected from overland storm flow. Some characteristics of the soils underlying the ponded pastures and the adjacent native pasture at Granite Vale are given in Table 1. Field procedure

Cattle were excluded from a 20 x 20 m area at each site. A 12 x 12 m square inside the protected area was divided into 36 plots, each 2 x 2 m. Four randomly 441

442

K. L. Weier et al. Table I. Characteristics

of the soil underlying

the ponded pasture species, Hymenachne

and A/man,

at the initial sampling Mineral

C:N ratio

N (pgg-‘)

type Sodic duplex (Dy 3.43)t

&5 5-10 l&l5

1.03 1.63 I .47

<4 <4 <4

0.23 0.05 0.05

3.85 0.81 0.68

16.7 16.2 13.6

0

Granite Vale Narhv

Sodic duplex (Dy 3.43)

&5 5-10 l&-l5

1.50 1.57 1.50

4 <4 <4

0.08 0.04 0.03

I .25 0.52 0.46

IS.6 13.0 15.3

0.9 0. I 0.1

3.6 1.9 2.5

Bear’s Lagoon

Black

(t5 5-10 l&l5

1.05 0.99 1.14

34 25 26

0.09 0.08 0.07

1.18 1.14 1.03

13.1 14.3 14.7

II.5 8.3 3.5

2.0 0.5 0.7

2k-man

(Up 5.13)

Bicarb.P

Total C (%)

Granite Vale H.vmenochne

Site

BD* (Mgm ‘)

Total N (%)

Depth (cm)

Soil

hg kg 9

NO,- -N 0 0

NHt-N II.0 1.2 1.1

*BD = bulk density. tNorthcote (1965).

selected plots were sampled every 6 wk for 48 wk for: (a) intact soil-plant cores by driving 16.5 x 12 cm dia PVC tubes into the soil; and (b) dry matter yield and N uptake by harvesting a 1 x 1 m quadrat. Each plot was sampled only once during the experiment. Four 9 x 9 cm nylon mesh bags containing 3 g dried Hymenachne or Aleman leaf material, and eight cellophane strips dyed with Remazol Blue (Moore et aI., 1979), were placed beneath the litter surface at each site to measure organic matter decomposition over 6 and 2 wk, respectively.

streaked to full strength potato-sucrose-malic acid agar prior to storage on glass beads at -70°C. Acetylene reduction measurements were performed directly in Wheaton bottles fitted with rubber seals. Air (12%) was replaced with C2H2 and C2H4 production measured using the procedure of Weier (1980a). Identification of isolates was based on morphology, biochemical tests and the use of API 20E strips (Bio Merieux SA, France).

RESULTS

Laboratory procedure

The soil-plant cores were kept for 24 h at 25°C in sealed perspex chambers in which 10% of the air had been replaced by acetylene (CzHz). Ethylene (CZH4) production was measured by gas chromatography on a 1 cm3 sample removed from the headspace of each chamber after 24 h (Weier, 1980a). Mineral N was extracted with 2 M KC1 (Catchpoole and Weier, 1980) and NO,- -N and NHJ-N concentration determined calorimetrically (Henzell et al., 1968). Total N was estimated by Kjeldahl digestion of ground material (Dalal et al., 1984) total C by dry combustion (Carr, 1973) and available P by extraction with bicarbonate and calorimetric determination (Colwell, 1965). Leaf material bags were washed and oven-dried prior to weighing. Cellophane decomposition from the buried strips was measured using the dye-release technique of Moore et al. (1979). Isolation of putative NJ-jxing bacteria

Bacterial isolates were obtained from adventitious roots and washed root pieces selected from the soil-plant cores that showed the greatest C2Hz reduction. Root pieces were cut into 1 cm segments, placed on or just below the surface of N-free semi-solid malic acid medium (pH adjusted to 6.8) and then incubated for 72 h at 32°C (Dobereiner, 1992). When pellicles formed at or near the surface, or when the bromothymol indicator turned blue, cultures were streaked to one-fifth strength potato-sucrose-malic acid agar. Single colony isolates were reinoculated to the N-free semi-solid medium and again incubated for 72 h at 32°C. Putative Nz-fixing species (pellicle formers or metabolically-active cultures) were

The initial soil cores at Granite Vale contained undetectable or very low concentrations of NO<-N while NH:-N was concentrated in the top 5 cm of soil (Table 1). At Bear’s Lagoon, NO<-N was concentrated in the top 1Ocm of soil and NH:-N concentrations were low. The total N content of the top 5 cm of soil under ponds at Granite Vale was three times larger than that found for dryland native pasture. The higher value under ponds was associated with a 2.5 cm layer of organic matter on the soil surface which was reflected in the higher total C content for Granite Vale. No equivalent organic matter layer was evident or expected in the newly planted pasture at Bear’s Lagoon. Available P at Granite Vale was seven times lower than found at Bear’s Lagoon where the concentrations were considered sufficient for pasture growth. N2-ase activity was detected in intact cores of the Aleman pasture within 2 months of planting with mean values at different sampling times ranging from 76 to 380 g N ha - ’d - ’(Fig. 1). Nz-fixation rates were calculated by using a ratio of 3 mol CzH2 reduced per mol of N2 fixed. Endogenous CzH4 production from ungassed cores was not measured as Weier (1980a) found no significant amounts of C2H4 present in the headspace above intact soil-plant cores of other tropical grasses. The greatest NI-ase activity occurred at the end of February 1993 following several cycles of flooding and draining of the pasture and accounted for 48% of the N accumulated by the grass. For the Hymenachne soil-plant cores, there was a steady decrease in Nz-ase activity from the maximum value of 179gNha-‘d-‘inMay1992toSgNha-‘d-’in April 1993, despite the rapid increase in biomass

N&xation

443

in ponded grass pastures 0

400

??

Hymenachne tops Hymenachne litter

A Aleman

0

2 3 4 1418 2419 12/l 92 93 92

1

4/s 92

s 2512 93

6 l/4 93

7

tops

4 S 6 1 2 3 1418 2419 22/10 12/l 2512 l/4 93 93 93 92 92 92

0

Sampling time (date)

7 13/S 93

8

Sampling time (date)

Fig. 1. N2-ase activity (g N ha-l d-l), as measured by the CZHZ reduction assay, of intact soil plus plant cores of the ponded pasture grasses Hymenachne and Aleman, sampled during 1992-1993 from Granite Vale and Bear’s Lagoon, respectively. Vertical bars show SEs.

Fig. 2. Dry matter yield during 1992-1993 from sequential cuts of the ponded pasture grasses, Hymenachneand Aleman. Vertical bars show SEs.

a lack of soil water. The quantity production following the inundation of the pasture in early January 1993 (Fig. 2). Nrase activity was also found on adventitious roots of Hymenachne which were produced on submerged stem nodes following

flooding of the pasture. A maximum Nz-fixation rate of 244 PM C2H4 per mg root fresh wt h- ’ (mean value = 54) was found on fibrous, brown roots with multiple short lateral roots extending from the node closest to the surface. Bacteria were isolated from these root pieces but await identification. Some formed pellicles when grown in semi-solid media but have not been identified. However, bacteria isolated from roots from soil-plant cores during a preliminary study were identified as Enterobacter agglomerans, Klebsiella oxytoca, Pseudomonas paucimobilis and Agrobacterium tumefaciaens. None of these isolates produced pellicles characteristic of the genus Azospirillum. Biomass production in the Aleman pasture was stimulated by the flooding, draining and grazing cycles imposed from August to December 1992. Maximum dry matter yields of 6.5 and 9.4 t ha - ’occurred in May, 1993 for the Hymenachne and Aleman pastures, respectively (Fig. 2). However, the percentage of crude protein of both grass species decreased as the age of the pasture increased (Table 2). The highest crude protein value of 15.5% was found in the Hymenachne leaves when the growth of the pasture was restricted through

of leaf litter recovered from the Hymenachne pasture at the end of winter ranged from 3.8 to 5.5 t ha-’ and decreased once flooding of the pasture occurred in early January 1993 (Fig. 2). Crude protein content of the leaf litter was higher than that found for Hymenachne green stems (Table 2). The decomposition rates measured using the dyed cellophane strips and the leaf bags were correlated with available water (Table 3). Pumping was curtailed from November 1992 at Bear’s Lagoon due to severe drought and subsequent decomposition was small. However, summer rains in early January 1993 at Granite Vale substantially accelerated decomposition of the leaf litter material. Because of the difficulties experienced with the removal of silt from leaf litter bags at Bear’s Lagoon, decomposition rates were calculated from cellophane strips only. DISCUSSION The Granite Vale soil was deficient in both P and N, and it is possible that the plants obtained some of their nutrient requirements from other sources. Resorption of P by the clay particles may negate the expected release of P from soil sediments under submerged conditions (Ponnamperuma, 1972). The low N1-ase

activities recorded both before and during the rapid growth phase of Hymenachne after inundation is in

Table 2. Percent N, crude protein and N accumulatedin the above-ground herbage of the ponded pasture grasses, Hymemtchne during

the 9-month

sampling

period at Granite

Vale and Bear’s Lagoon,

E. polystachya

H. amplexicaulis Sampling date

Green leaf %N C.P.’

14-8-92 24-9-92 22-10-92 12-I-93 25-2-93 1493 13-5-93

2.5 ND 1.4 ND ND ND ND

Green stem C.P. %N

IS.5

0.5

ND 9.0 ND ND ND ND

ND 0.4 ND ND ND ND

*C.P. = %crude protein. ND, not determined.

2.9 ND 2.6 ND ND ND ND

and Alemon,

respectively

%N

Litter C.P.

%N

Tops C.P. kgN

0.8 ND 0.6 ND ND ND ND

:A 3.6 ND ND ND ND

ND ND ND 1.7 1.0 0.6 0.5

ND ND ND 10.4 6.4 4.0 2.9

ha-’

ND ND ND 15.0 29.7 39.6 29.4

Green leaf %N C.P.

%N

Tops C.P. kg N ha-’

ND ND ND ND 0.9 ND ND

ND 1.3 1.0 0.9 0.5 0.4 0.3

ND 1.9 6.1 5.7 2.8 2.3 1.6

ND ND ND ND 5.3 ND ND

ND 6.4 7.0 15.2 33.1 28.5 22.6

K. L. Weier et al.

444

Table 3. Percentage decomposition of leaf litter bags buried for 6 weeks and cellophane strips buried for 2 weeks beneath the Hymenachne and Aleman pastures at Granite Vale and Bear’s Lagoon. Values are the means of four litter bags and eight cellophane strips and werecalculated after transforming the percentages using the arcsine function Sampling (burial) date 14-8-92 24-9-92 24- 1O-92 2-12-92 14-I-93 25-2-93 l-4-93

% Cellophane

decomposition*

Bear’s Lagoon

Granite

36.6 + 26.6 + Nil 1.3 + 6.9 f 8.7 + 3.4 f

5.5 8.8 1.8 12.8 19.2 5.3

10.7 + NA 0.5 * Nil 47.0 * 30.9 f 9.7 f

Vale 13.1 0.9 3.9 8.6 4.7

% Leaf decomposition Granite Vale NAt NA 12.8 +_ 2.8 16.3 f 3.0 55.7 f I.1 44.5 + 3.3 28.0 ? 1.4

*Mean -+ SE. tNot available.

conflict with the high crude protein contents measured in the tops. Apparently, the rapid decomposition of the organic matter layer following inundation at the Granite Vale site released sufficient mineralized N and P for plant growth, and fixed N was less important. At Bear’s Lagoon, neither P nor N limited the growth of the Aleman pasture. However, biologically fixed N was estimated to form a significant amount of the N accumulated by the plant tops and represented about half of the N required for plant growth. For both ponded pasture species, the N accumulated by the plant tops was greater than that found for dryland species growing in similar soil (Weier and Pittaway, 1993). Live-weight gains of yearling steers and bullocks on ponded pastures at Granite Vale had been recorded prior to the experimental period. Animal performance over the seasonally dry winter period (March-July) in 1988 was exceptional with yearlings gaining 53 kg ha- ’and bullocks 38 kg ha - ’despite poor growing conditions over the previous autumn due to excess rain “drowning” the grasses where ingress of water was too rapid (Wildin, 1993). This compared with a normal production capacity of 20-40 kg liveweight gain ha - ’ y - I for steers grazing native dryland pasture in Queensland (Middleton et al., 1991). The microbial decomposition rates of the leaf litter at Granite Vale were very high, and were enhanced by inundation after summer rainfall. Decomposition under anaerobic conditions requires less N than under aerobic conditions with about 0.5% N required for the decomposition to result in net N mineralization (Patrick, 1982). At Granite Vale, ca. 9 kg ha- ’ of inorganic N could be expected to be released from the litter and available for growth of the Hymenachne pasture. This is equivalent to 23% of the maximum yield of N in the pasture. More N release could be anticipated through mineralization of the organic matter layer because of its low C-to-N ratio. The decomposition rates recorded here exceeded those found for the temperate macrophyte Ludwigiu peptoides, recognized as a highly efficient nutrient harvester (Rejmankova, 1992). The pasture at Bear’s Lagoon lacked a litter layer, but the high decompo-

sition rates recorded during the flooding cycles of August and September 1992 indicated the potential for enhanced productivity via mineralization once the litter layer developed. Under inundation, Hymenachne was capable of extremely rapid stem internode elongation, stem DM production and nodal adventitious root production. The highest rates of Nz-ase activity were recorded from these adventitious roots, suggesting that associative Nz-fixation was tightly coupled with photosynthesis, as observed in the paspalum (Dobereiner et al., 1972b) and spartina (Whiting et al., 1986) diazotrophic systems. The Nz-ase activities were considerably less than the specific activities observed for nodules of Vigna (Lawn and Bushby, 1982) but higher than the rates observed for incubated roots of dryland tropical grasses growing at Beerwah in SE Queensland (Weier, 1980b). The inability to isolate Azospirillum strains from roots from intact soil-plant cores of both grasses during the preliminary study could be because the bacteria were either absent or that the conditions for growth during isolation were unfavourable (H. J. Woods, pers. commun.). However, during the seasonal study using more dilute media, pellicle formers were isolated from root pieces of both grasses perhaps indicative of Azospirillum strains. A more detailed study of the isolates is required to assess the dynamics of N2-fixing bacteria populations associated with the roots of ponded pasture grass species. For the initial growth of the Aleman pasture, N,-fixation appeared to be the primary pathway through which N was accumulated by the plant. However, as pastures age, plant debris will increase, with N mineralization assuming a more important role in increasing plant protein and productivity of the pasture. For the older Hymenachne pasture, contributions from N*-fixation were smaller and litter decomposition and N mineralization appeared to be the major avenues for maintaining productivity. Nz-fixation may have played a larger role during the establishment phase of the Hymenachne pasture. Acknowledgements-We

thank

MS Helen

Woods

for her

assistance with bacterial isolations and identifications during this study. The financial support of the University of Queensland Foundation Ltd is also gratefully acknowledged. REFERENCES

App A., Santiago T., Daez C., Menquito C., Ventura W., Tirol A., POJ., Watanabe I., de Datta S. K. and Roger P. (1984)Estimation of the nitrogen balance for irrigated rice and the contribution of phototrophic Field Crops Research 9, 17-27.

nitrogen

fixation.

Buresh R. J., Casselman M. E. and Patrick W. H. (1980) Nitrogen fixation in flooded soil systems, a review. In Advances in Agronomy (N. C. Brady, Ed.). Vol.33. pp. 149-192. Academic Press, New York. ’ Carr C. E. (1973) Gravimetric determination of soil carbon using the’ Leco induction furnace. Journal of Science of Food and Agriculrure 24, 1091-1095.

445

N1-fixation in ponlded grass pastures Catchpoole V. R. and Weier K. L. (1980) Water pretreatment helps during extraction of mineral-N from a clay soil. Communications of Soil Science and Plant Analysis 11, 327-333. Colwell J. D. (1965) An automated procedure for the determination of phosphorus in sodium hydrogen carbonate extracts of soils. Chemistry and Industry 22 May, 893-895. Dalal R. C., Sahrawat K. L. and Myers R. J. K. (1984) Inclusion of nitrate and nitrite in the Kjeldahl nitrogen determination of soils and plant materials using sodium thiosulphate. Communications of Soil Science and Plant Analysis 15, 1453-1461. Dobereiner J. (1992) The genera Azospirillum and Herbaspirillwn. In The Prokarjotes, 2nd E&I (A. Balows, H. G.Tnmer. M. Dworkin. W. Harder and K. H.Schleifer. Eds), pp.-103-l 16. Springer-Verlag, New York. Dobereiner J., Day J. M. and Dart P. J. (1972a) Nitrogenase activity in the rhizosphere of sugar cane and some other tropical grasses. Planr and Soil 37, 191-196. Dobereiner J., Day J. M. and Dart P. J. (1972b) Nitrogen activity and oxygen sensitivity of the Paspalum notatumAzotobacter paspali association. Journal of General Microbiology 71, 103-l 16. H-11 E. F., Vallis I. and Lindquist J. K. (1968) Automatic calorimetric methods for the determination of nitrogen in digests and extracts of soil. Transactions 9th International Congress of Soil Science, pp. 513-520. Adelaide. Lawn R. J. and Bushby H. V. A. (1982) Effect of root, shoot and Rhizobium strain on nitrogen fixation in four asiatic Vigna species. New Phytologist 92,425-434. Middleton C., Murphy K. and Wildin J. H. (1991) Productivity of ponded pastures In Probing Ponded Pastures (Qld Department Primary Industries, Ed.), Sect. 2, pp. l-8. University of Central Queensland, Rockhampton, 16-18 July 1991. Moore R. L., Basset B. B. and Swift M. J. (1979) Developments in the Rem-1 Brilliant Blue dye-assay for studying the ecology of cellulose decomposition. Soil Biology & Biochemistry 11, 311-312.

Northcote K. H. (1965) CSIRO Australian Division of Soils, Divisional Report No. 2/65. Patrick W. H. (1982) Nitrogen transformations in flooded soils. In Nitrogen in Agricultural Soils (F. J. Stevenson, Ed.), pp. 449-465. Monograph No. 22, American Society of Agronomy, Madison, WI. Ponnamperuma F. N. (1972) The chemistry of submerged soils. In Advances in Agronomy (N. C.Brady. Ed.). pp. 29-96. Academic Press, New York. Rejmankova E. (1992) Ecology of creeping macrophytcs with special reference to Ludwigia peploides (H.B.K.) Raven. Aquatic Botany 43, 283-299.

Weier K. L. (1980a) Nitrogenase activity associated with three tropical grasses growing in undisturbed soil cores. Soil Biology & Biochemistry 12, 131-136. Weier K. L. (1980b) Nitrogen fixation associated with grasses. Tropical Grasslands 14, 194-201. Weier K. L. and MacRae I. C. (1980) Nitrogenase activity in grass-bacteria associations in Northern Australia. In Current Perspectives in Nitrogen Fixation (A. H. Gibson and W. E. Newton, Eds), p.491. Australian Academy of Science, Canberra. Weier K. L. and Pittaway P. A. (1993) Nitrogen economy of ponded pasture systems. In Improving Beef Production with Better Port&d Pastures (J. H. Wildin, Ed.), pp. 109-112. Rockhampton, 2@-23 April. Whiting G. J., Candy E. L. and Yoch D. C. (1986) Tight coupling of root-associated nitrogen fixation and plant photosynthesis in the salt marsh grass Spartina alternipora and carbon dioxide enhancement of nitrogenase activity. Applied and Environmental Microbiology 52, 108-l 13. Wildin J. H. (1993) Ponded pasture systems for beef production in the dry seasons of northern Australia. In Proceedings

XVII

Znternarional

Grasslands

Congress,

Session 13, Palmerston North. New Zealand. Februarv.<, 1993. Wildin J. H. and Chapman D. G. (1988) Ponded pasture systems-capitalising on available water. Queensland Department of Primary Industries, Rockhampton, RQR 87006.